JP2019519217A5

JP2019519217A5 -

Info

Publication number: JP2019519217A5
Application number: JP2018560605A
Authority: JP
Filing date: 2017-05-04
Publication date: 2020-06-11
Anticipated expiration: 2037-05-04

Description

他の実施形態
本発明は、その詳細な説明と共に説明してきたが、前述の説明は、添付の特許請求の範囲によって定義される本発明の範囲を説明するためのものであり、それを制限するためのものではないと理解される。他の態様、利点、および変更は、以下の特許請求の範囲の範囲内である。
本発明の様々な実施形態を以下に示す。
１．バイオ人工肺臓器を提供する方法であって、
ヒトドナーから増殖性の基底幹細胞集団を提供するステップであって、前記細胞が、好ましくは前記ドナーの気道から得たＫｒｔ５+ｐ６３+細胞である、ステップと；
任意選択でＲＯＣＫ阻害剤の非存在下で、任意選択で最大５回の継代（好ましくは、細胞を６０〜１００％、好ましくは８０％コンフルエンシーで継代した）まで、前記細胞を培養において維持および増大させるステップと；
気道および実質的な血管系を含む（無細胞）肺組織マトリックスを提供するステップと；
前記肺組織マトリックスに、前記気道を通して前記幹細胞を、および前記血管系を通して内皮細胞を播種するステップと；
前記気道における機能的上皮および機能的血管系の形成にとって十分な条件で前記マトリックスを維持するステップであって、前記マトリックスの維持が、ｎｏｔｃｈ阻害剤、好ましくはガンマセクレターゼ阻害剤を含む液体培地を使用して、第１の所望の程度に臓器を成熟させて、湿潤成熟臓器を産生させるために十分な時間、前記肺組織マトリックスに湿式換気を提供することと、任意選択で湿式換気の間、臓器チャンバーにおいて実質的に一定の流体レベルを維持することとを含む、ステップと、
を含む方法。
２．前記臓器チャンバーが、各々がチャンバー圧センサーによって伝達されるデータに反応して二方向排液ポンプを制御する制御モジュールによって制御されるチャンバー圧センサーと二方向排液チャンバーポンプとを備える、上記１に記載の方法。
３．静脈ラインにおける圧力レベルを培地リザーバーにおける圧力レベルと平衡にすることによって、肺内外圧差を防止するステップをさらに含む、上記１に記載の方法。
４．前記臓器チャンバーが、前記臓器チャンバーに接続された空気圧制御モジュールをさらに備え、前記空気圧制御モジュールが、
吸気相の間、前記臓器チャンバーにおいて陰圧を生成し；
平衡相の間、前記臓器チャンバー圧を維持し；および
呼気相の間、前記臓器チャンバーにおいて陽圧を生成する、
上記１に記載の方法。
５．湿式換気が、
コントローラーに接続された二方向気管ポンプを含む気管ラインを、培地リザーバーに接続するステップと；
前記二方向気管ポンプを使用して前記肺組織マトリックスを培地によって膨らませるステップと；
前記二方向気管ポンプを使用し前記肺組織マトリックスから培地を引き抜いて、前記肺組織マトリックスをしぼませるステップとを含み、
前記培地が湿式換気の間、絶えず補充される、
上記１に記載の方法。
６．前記湿式換気が、
各々がコントローラーに接続された第１のポンプと第２のポンプとを含む前記気管ラインを、培地リザーバーに接続するステップと；
前記第１のポンプを使用して、前記肺組織マトリックスを培地によって膨らませるステップと；
前記第２のポンプを使用し前記肺組織マトリックスから培地を引き抜いて、前記肺組織マトリックスをしぼませるステップとを含み、
前記培地が湿式換気の間、絶えず補充される、
上記１に記載の方法。
７．前記コントローラーが、前記気管ラインに接続された気管圧センサーによって伝達されるデータに反応して前記二方向気管ポンプを制御する、上記６に記載の方法。
８．ｎｏｔｃｈ阻害剤を含む液体培地を使用して湿式換気を少なくとも２、５、７、または１０日間提供し、任意選択で、その後に、ｎｏｔｃｈ阻害剤を含まない液体培地を使用して追加の湿式換気をするステップを含む、上記１に記載の方法。
９．前記肺組織マトリックスが、外から添加したテネイシン−ｃおよび／または外から添加したフィブリリン−２の１つまたは両方を含む、上記１に記載の方法。
１０．播種の前に、前記肺組織マトリックスにテネイシン−ｃまたはフィブリリン−２の１つまたは両方を接触させるステップを含む、上記９に記載の方法。
１１．上記１〜１０に記載の方法によって産生された機能的な肺。
１２．前記臓器が、完全な肺またはその血管柄付きの部分である、上記１１に記載の機能的な肺。
１３．上記１１に記載の肺を対象に移植するステップを含む、肺活量が損なわれたまたは低減した対象を治療する方法。
１４．肺活量が損なわれたまたは低減した対象を治療する方法における、上記１１に記載の機能的な肺の使用。
１５．肺活量が損なわれたまたは低減した対象を治療する方法に使用するための上記１１に記載の機能的な肺。 Other Embodiments While the present invention has been described in conjunction with its detailed description, the foregoing description is for the purpose of illustrating and limiting the scope of the invention as defined by the appended claims. It is understood that it is not intended. Other aspects, advantages, and modifications are within the scope of the following claims.
Various embodiments of the present invention are shown below.
1. A method of providing a bioartificial lung organ, comprising:
Providing a proliferative basal stem cell population from a human donor, said cells being Krt5+p63+ cells, preferably obtained from the respiratory tract of said donor.
The cells are in culture, optionally in the absence of a ROCK inhibitor, optionally up to a maximum of 5 passages (preferably 60-100%, preferably 80% confluency). Maintaining and augmenting;
Providing a (cellular) lung tissue matrix comprising airways and a substantial vasculature;
Seeding the lung tissue matrix with the stem cells through the airways and endothelial cells through the vasculature;
Maintaining said matrix in conditions sufficient for the formation of functional epithelium and functional vasculature in said airways, said matrix being maintained using a liquid medium comprising a notch inhibitor, preferably a gamma secretase inhibitor. And providing the wet ventilation to the lung tissue matrix for a time sufficient to mature the organ to a first desired degree and produce a wet mature organ, and optionally during the wet ventilation. Maintaining a substantially constant fluid level in the chamber;
Including the method.
2. 1 wherein said organ chamber comprises a chamber pressure sensor and a two-way drainage chamber pump each controlled by a control module controlling a two-way drainage pump in response to data transmitted by the chamber pressure sensor. The method described.
3. The method of claim 1 further comprising the step of preventing transpulmonary pressure differential by balancing the pressure level in the venous line with the pressure level in the media reservoir.
4. The organ chamber further comprises a pneumatic control module connected to the organ chamber, the pneumatic control module,
Creating a negative pressure in the organ chamber during the inspiration phase;
Maintaining the organ chamber pressure during the equilibrium phase; and
Generate positive pressure in the organ chamber during the expiratory phase,
The method described in 1 above.
5. Wet ventilation
Connecting a tracheal line containing a two-way tracheal pump connected to a controller to the medium reservoir;
Inflating the lung tissue matrix with medium using the two-way tracheal pump;
Withdrawing medium from the lung tissue matrix using the two-way tracheal pump to deflate the lung tissue matrix,
The medium is constantly replenished during wet ventilation,
The method described in 1 above.
6. The wet ventilation is
Connecting the tracheal line to a culture medium reservoir, the tracheal line comprising a first pump and a second pump each connected to a controller;
Inflating the lung tissue matrix with a medium using the first pump;
Withdrawing medium from the lung tissue matrix using the second pump to deflate the lung tissue matrix,
The medium is constantly replenished during wet ventilation,
The method described in 1 above.
7. 7. The method according to claim 6, wherein the controller controls the bidirectional tracheal pump in response to data transmitted by a tracheal pressure sensor connected to the tracheal line.
8. Providing wet ventilation using a liquid medium containing a notch inhibitor for at least 2, 5, 7, or 10 days, optionally followed by additional wet ventilation using a liquid medium containing no notch inhibitor. The method according to 1 above, which comprises the step of:
9. The method of claim 1 wherein the lung tissue matrix comprises one or both of exogenously added tenascin-c and/or exogenously added fibrillin-2.
10. 10. The method of claim 9, comprising contacting the lung tissue matrix with one or both of tenascin-c or fibrillin-2 prior to seeding.
11. A functional lung produced by the method according to 1 to 10 above.
12. 12. The functional lung according to 11 above, wherein the organ is a complete lung or a vascularized portion thereof.
13. 13. A method of treating a subject with impaired or reduced vital capacity, comprising the step of transplanting the lung of claim 11 to the subject.
14. The use of the functional lung according to the above 11, in a method of treating a subject with impaired or reduced vital capacity.
15. A functional lung according to claim 11 for use in a method of treating a subject with impaired or reduced vital capacity.

Claims

A method of providing a bioartificial lung organ, comprising:
Providing a proliferative basal stem cell population from a human donor, said cells being Krt5+p63+ cells;
Passaging of up 5 times, a step of maintaining and increasing in culturing the cells, the steps the cells are passaged at 60% to 100% confluency;
Comprising: providing a cell-free lung tissue matrix including airway and vascular system, the lung tissue matrix comprises one or both of fibrillin -2 added from tenascin -c or outer exogenously added Steps ;
Seeding the lung tissue matrix with the stem cells through the airways and endothelial cells through the vasculature;
using a liquid medium containing n Otch inhibitor for a time sufficient to mature organ to the degree that is preselected, the matrix organ chamber under conditions comprising providing a wet ventilation to the lung tissue matrix Steps to maintain ,
Including the method.

2. The organ chamber comprises a chamber pressure sensor and a two-way drainage chamber pump each controlled by a control module that controls the two-way drainage pump in response to data transmitted by the chamber pressure sensor. The method described in.

The method of claim 1, further comprising preventing transpulmonary pressure differentials by balancing the pressure level in the venous line connected to the pulmonary vein of the pulmonary tissue matrix with the pressure level in the media reservoir.

The organ chamber further comprises a pneumatic control module connected to the organ chamber, the pneumatic control module,
Creating a negative pressure in the organ chamber during the inspiration phase;
Maintain the organ chamber pressure during the equilibrium phase; and generate positive pressure in the organ chamber during the expiratory phase,
The method of claim 1.

Wet ventilation
Wherein a lung tissue matrix trachea lines to the connected airway, the trachea lines containing bidirectional trachea pump connected to the controller, and connecting the medium reservoir;
Inflating the lung tissue matrix with medium using the two-way tracheal pump;
Withdrawing culture medium from the lung tissue matrix using the two-way tracheal pump to deflate the lung tissue matrix,
The medium is constantly replenished during wet ventilation,
The method of claim 1.

The wet ventilation is
Connecting a tracheal line also to the airway of the lung tissue matrix, the tracheal line comprising a first pump and a second pump , each connected to a controller, to a culture medium reservoir;
Inflating the lung tissue matrix with a medium using the first pump;
Withdrawing medium from the lung tissue matrix using the second pump to deflate the lung tissue matrix,
The medium is constantly replenished during wet ventilation,
The method of claim 1.

7. The method of claim 6, wherein the controller controls the two-way tracheal pump in response to data transmitted by a tracheal pressure sensor connected to the tracheal line.

Providing wet ventilation using a liquid medium containing a notch inhibitor for at least 2, 5, 7, or 10 days, optionally followed by additional wet ventilation using a liquid medium containing no notch inhibitor. The method of claim 1 including the step of:

2. The method of claim 1 , comprising contacting the lung tissue matrix with one or both of tenascin-c or fibrillin-2 prior to seeding.

A functional lung produced by the method of claim 1 .

11. The functional lung of claim 10 , wherein the organ is a complete lung or vascularized portion thereof.

Lung claim 10 for use in a method of treating a subject pulmonary activity of the was impaired or reduced.

The method of claim 1, wherein the Krt5 + p63 + cells are obtained from the airways of the donor.

The method of claim 1, wherein the cells are passaged at 80% confluency.

The method of claim 1, wherein the notch inhibitor is a gamma secretase inhibitor.

The method of claim 1, wherein the cells are passaged in culture in the absence of a ROCK inhibitor prior to seeding into lung tissue matrix.